Department of Energy–Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

(B) Rapid assay for MeJA-induced PPO activity in tomato leaves. Leaf juice from individual leaflets of 15-day-old plants was expressed on a nitrocellulose membrane and assayed for PPO activity as described by Howe and Ryan (1999). Wild-type plants were treated with MeJA (+) or ethanol (−) as a control. F2 plants derived from a cross between jai1-1 and the wild type were treated with MeJA before testing for PPO activity. Dark brown staining indicates the presence of PPO activity.

(C) Response of germinating seedlings to MeJA. Wild-type, heterozygous (J/j), and homozygous (j/j) seeds were germinated on moist filter paper and exposed to MeJA (+) for 2 days. A wild-type seedling grown in the absence of MeJA (−) is shown as a control.

(D) and (E) Scanning electron micrographs of the surface of developing green fruit from wild-type (D) and jai1-1(E) plants. Arrows in (D) denote type-VI (t-VI) and type-I (t-I) trichomes. The tissue fixation procedure used for scanning electron microscopy affected the structure of type-I trichomes, which were 1 to 2.5 mm long on intact tissue (see [A] and [D]).

Three-week-old Micro-Tom and jai1-1 plants were exposed to MeJA vapor for various lengths of time (hours) in an enclosed box. Leaves from similarly treated plants of the same genotype were pooled for RNA extraction. RNA isolated from untreated plants (0-h time point) also was analyzed as a control. RNA gel blots were hybridized to cDNA probes representing four late genes (PI-I, PI-II, CATHEPSIN D INHIBITOR [CDI], and THREONINE DEAMINASE [TD]) and four early genes (LIPOXYGENASE D [LoxD], ALLENE OXIDE SYNTHASE2 [AOS2], OPDA REDUCTASE3 [OPR3], and PROSYSTEMIN [PSYS]). Blots also were hybridized to the LeCoi1 cDNA and, as a loading control, to an eIF4A probe.

Resistance of jai1-1 Plants to the Two-Spotted Spider Mite Is Severely Compromised.

(A) to (D) Fifteen adult female mites were transferred to a single leaf on 3-week-old wild-type ([A] and [C]) and jai1-1 ([B] and [D]) plants. Photographs of the infested leaves were taken 6 days ([A] and [B]) and 11 days ([C] and [D]) after challenge. The boxed area in (B) shows the initial effects of feeding damage on jai1-1.

(E) and (F) Photographs of representative 7-week-old wild-type (E) and jai1-1(F) plants at 30 days after infestation.

(G) Two-choice assay to measure the preference of spider mites for wild-type (WT) or jai1-1 plants. Ten adult female spider mites were placed in an arena equidistant from wild-type and jai1-1 leaflets. The number of mites that moved to one or the other of the leaflets was determined 1 h after initiating the assay, as was the number of mites that failed to make a choice (nc). Data represent means and standard errors from 16 repetitions (total of 160 mites).

Deduced Amino Acid Sequence of LeCOI1 Compared with Homologs in Arabidopsis and Rice.

Sequence alignments were performed using the GCG sequence analysis package (Genetics Computer Group, Madison, WI). Amino acids that are either identical or similar between the three sequences are indicated in black and gray, respectively. Arrows denote the approximate positions of the 16 imperfect LRR domains. The arrow and LRR domain number are indicated beneath the corresponding LRR. The asterisk denotes the Gly residue that is changed to a Cys in jai1-2 plants. GenBank accession numbers are given at the end of Methods. The N-terminal end of the rice sequence contains 35 additional amino acids that are not included in the alignment.

(A) Scheme of a 9.4-kb region of genomic DNA containing LeCoi1 and a gene encoding a putative MYB transcription factor (pMYB). The three exons (E1 to E3) and intervening sequences that constitute LeCoi1 are drawn to scale. Only the translated portion of the first and last exons are shown, together with the start (ATG) and stop (TAG) codons within the respective exons. The 6.2-kb region of DNA that is deleted in jai1-1 plants is shown by the horizontal hatched line. B indicates BglII restriction sites used for the DNA gel blot analysis shown in (C), and B* indicates the presence of two BglII sites that are separated by 24 bp. cDNA probes used to detect the 5′ and 3′ ends of the gene in (C) are shown as thick horizontal bars.

(B) Structures of the Arabidopsis (AtCOI1) and rice (OsCOI1) homologous genes drawn to the same scale as LeCoi1 in (A).

(C) DNA gel blot analysis of BglII-restricted genomic DNA from the wild type (lanes 1), jai1-1 (lanes 2), and an F1 hybrid produced from a cross between the wild type and jai1-1 (lanes 3). Three identical blots were hybridized to a full-length (FL) LeCoi1 cDNA probe or to probes corresponding to the 5′ and 3′ ends of the cDNA (see [A]). Numbers at left indicate the positions of DNA size standards (in kb).

(D) RNA gel blot analysis of LeCoi1 transcript levels in wild-type (WT) and jai1-1 plants. Five micrograms of total RNA from root (R), petiole (P), leaf (L), unopened flower bud (F), sepal (S), and immature green fruit (G) was immobilized to a membrane and hybridized to a LeCoi1 cDNA probe (top gel). A duplicate blot was hybridized to an eIF4A cDNA probe as a loading control.

Total RNA was prepared from wild-type (WT), jai1-1 (jai1), and T2 siblings from two 35S-Coi1–complemented lines (T2-08 and T2-13) either before (−) or after (+) exposure to MeJA vapor for 12 h. For each transgenic line tested, a PCR assay was used to identify siblings that either harbor (+) or lack (−) the 35S-LeCoi1 transgene. RNA gel blots were hybridized to cDNA probes for PI-II and LeCoi1 and to eIF4A as a loading control.

↵a Expression was not detectable, indicating that the microarray signal was below background levels.

Three-week-old Castlemart (wild-type) and jai1-1 plants were treated with either MeJA or ethanol (mock control) for 8 h. Leaf tissue was harvested for RNA isolation after the treatment. A custom cDNA microarray slide representing ∼500 tomato genes was hybridized simultaneously to probes derived from RNA isolated from ethanol- and MeJA-treated plants of the same genotype (i.e., wild type or jai1-1). Numbers represent the mean expression ratio (MeJA:ethanol) of two independent biological replicates for each experiment. Genes (GenBank accession number and putative function) that were differentially regulated by >2.5-fold in response to MeJA in wild-type plants are listed.